RNAI induced huntingtin gene suppression

ABSTRACT

The present invention provides for a double stranded RNA comprising a first RNA sequence and a second RNA sequence wherein the first and second RNA sequence are substantially complementary, wherein the first RNA sequence has a sequence length of at least 19 nucleotides and is substantially complementary to SEQ ID NO. 1. Said double stranded RNA is for use in inducing RNAi against Huntingtin exon 1 sequences. The double stranded RNA of to the invention was capable of reducing neuronal cell death and huntingtin aggregates in an animal model.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Phase of International PatentApplication No. PCT/EP2015/081157, filed Dec. 23, 2015, published onJun. 30, 2016 as WO 2016/102664 A1, which claims priority to EuropeanPatent Application No. 14200308.6, filed Dec. 24, 2014. The contents ofthese applications are herein incorporated by reference in theirentirety.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 22, 2017, isnamed 100607-0300SequenceListing.txt and is 20 KB.

BACKGROUND

The huntingtin gene, also referred to as the HTT or HD (Huntington'sdisease) gene, encodes for the huntingtin protein. The huntingtin geneis a large gene of about 13.5 kb (huntingtin protein is about 350 kDa).Huntington's disease is a genetic neurodegenerative disorder caused by agenetic mutation in the huntingtin gene. The genetic mutation involves aDNA segment of the huntingtin gene known as the CAG trinucleotiderepeat. Normally, the CAG segment in the huntingtin gene of humans isrepeated multiple times, i.e. about 10-35 times. People that developHuntington's disease have an expansion of the number of CAG repeats inat least one allele. An affected person usually inherits the mutatedallele from one affected parent. In rare cases, an individual withHuntington's disease does not have a parent with the disorder (sporadicHD). People with 36 to 39 CAG repeats may develop signs and symptoms ofHuntington disease, while people with 40 or more repeats almost alwaysdevelop the disorder. The increase in the size of the CAG repeat leadsto the production of an elongated (mutated) huntingtin protein. Thisprotein is processed in the cell into smaller fragments that arecytotoxic and that accumulate and aggregate in neurons. This results inthe disruption of normal function and eventual death of neurons. This isthe process that occurs in the brain which underlies the signs andsymptoms of Huntington's disease.

RNA interference (RNAi) is a naturally occurring mechanism that involvessequence specific down regulation of mRNA. The down regulation of mRNAresults in a reduction of the amount of protein that is expressed. RNAinterference is triggered by double stranded RNA. One of the strands ofthe double stranded RNA is substantially or completely complementary toits target, the mRNA. This strand is termed the guide strand. Themechanism of RNA interference involves the incorporation of the guidestrand in the RNA-induced silencing complex (RISC). This complex is amultiple turnover complex that via complementary base paring binds toits target mRNA. Once bound to its target mRNA it can either cleave themRNA or reduce translation efficiency. RNA interference has since itsdiscovery been widely used to knock down specific target genes. Thetriggers for inducing RNA interference that have been employed involvethe use of siRNAs or shRNAs. In addition, molecules that can naturallytrigger RNAi, the so called miRNAs, have been used to make artificialmiRNAs that mimic their naturally occurring counterparts. Thesestrategies have in common that they provide for substantially doublestranded RNA molecules that are designed to target a gene of choice.RNAi based therapeutic approaches that utilise the sequence specificmodality of RNAi are under development and several are currently inclinical trials (see i.a. Davidson and McCray, Nature Reviews—Genetics,2011; Vol. 12; 329-340).

As Huntington's disease involves the expression of a mutant huntingtinprotein, the accumulation thereof leading to disease, RNA interferenceprovides for an opportunity to treat the disease as it can reduceexpression of the huntingtin gene. The paradigm underlying this approachinvolves a reduction of the mutant Htt protein to thereby reduce thetoxic effects resulting from the mutant Htt protein to achieve areduction and/or delay of Huntington's disease symptoms, or even toprevent Huntington's disease symptoms altogether. Targeting huntingtingene suppression has been hypothesized in the prior art, including thelisting of about two thousand of hypothetical siRNA target sequences(WO2005105995). Strategies to reduce huntingtin gene expression areknown in the art and involve the specific targeting of mutant huntingtingenes (e.g. US20090186410, US20110172291). Alternatively, RNAinterference has also been employed to target both mutant and non-mutantgenes (e.g. Rodriguez-Lebron et al., 2005, Mol Ther. Vol 12 No. 4:618-633; Franich et al., 2008, Mol Ther, Vol. 16 No. 5; 947-956; Drouetet al., 2009, Annals of Neurology; Vol. 65 No. 3; 276-285 and McBride etal. Mol Ther. 2011 December; 19(12):2152-62; US20080015158,WO2008134646). In the latter case, knockdown of the wild type Huntingtinprotein was shown not to have any apparent detrimental effects.

SUMMARY OF THE INVENTION

The present invention provides for a double stranded RNA comprising afirst RNA sequence and a second RNA sequence wherein the first andsecond RNA sequence are substantially complementary, wherein the firstRNA sequence has a sequence length of at least 19 nucleotides and issubstantially complementary to SEQ ID NO. 1. A large number of targetsequences were tested for effective knockdown of the huntingtin gene.The selected double stranded RNA of the current invention was found tobe effective in reducing huntingtin gene expression. Said doublestranded RNA when provided in a cell, either directly via transfectionor indirectly via delivery of DNA (e.g. transfection) or viavector-mediated expression upon which the said double stranded RNA canbe expressed, is capable of reducing expression of both a mutatedhuntingtin gene and a normal huntingtin gene. Furthermore, it was shownthat the double stranded RNA of the invention was capable of reducingtarget gene expression when provided either as an siRNA or in a miRNAscaffold. When tested in an animal model, it was shown that a doublestranded RNA according to the invention was capable of reducing neuronalcell death and huntingtin aggregates. The double stranded RNA asprovided in the current invention provides for an improvement ascompared to double stranded RNAs in the art targeting the huntingtingene, or provides for at least an alternative thereto.

The double stranded RNA according to the invention can be provided as ansiRNA, a shRNA, a pre-miRNA or pri-miRNA. Such double stranded RNAs maybe delivered to the target cells directly, e.g. via cellular uptakeusing e.g. transfection methods. Preferably, said delivery is achievedusing a gene therapy vector, wherein an expression cassette for thesiRNA, shRNA, pre-miRNA or pri-miRNA is included in a vector. This way,cells can be provided with a constant supply of double stranded RNA toachieve durable huntingtin gene suppression without requiring repeatedadministration. Preferably, the viral vector of choice is AAV5. Thecurrent invention thus also provides for the medical use of a doublestranded RNA according to the invention, such as the treatment orHuntington's disease, wherein such medical use may also comprise anexpression cassette or a viral vector, such as AAV5, capable ofexpressing the said double stranded RNA of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C. Human huntingtin (HTT) gene and target sequences. (A)Schematic of the human HTT gene (L27350.1) with CAG expansions (black)and target sequences for miH1-H21 (light grey) (B) Exon 1 RNA sequenceof the HTT gene (SEQ ID NO.2). The CAG repeat sequence is from nts.367-429. (C) Schematic of target sequences tested for exon 1 (H1,185-205; H2, 186-206; H3, 189-209; H4, 191-211; H5, 194-214; H6,196-216; H7, 250-270; H8, 261-281, H9, 310-330; H10, 311-331, H11,339-359, H12, 345-365, H13, 454-474; H14, 459-479; H15, 477-497; H16,486-506; H17, 492-512; H18, 498-518; H19, 549-569; H20, 557-577; H21,558-578, H1-H21 corresponding to SEQ ID NOs.23-43). The sequencesdepicted are DNA sequences. The numbers refer to the corresponding RNAnucleotide sequences in SEQ ID NO.2. The corresponding RNA targetsequences of SEQ ID NO.2 have the sequence as listed in C) except thatwherein the DNA encodes a “t” the RNA encodes a “U”.

FIGS. 2A-2H. Examples of double stranded RNAs and expression cassettes.(A) Examples of pri-/pre-miRNA scaffold for miH12 pre-miH12-155 (SEQ IDNO.44) and pre-miH12-451 scaffold (SEQ ID NO.45) with miH12 guide (grey)indicated. (B) Schematic outlining of the double stranded RNAs inaccordance with the invention and how they can be processed by the RNAimachinery. A double stranded RNA may be a short hairpin RNA (1) or anextended siRNA (2). The hairpin RNA or extended siRNA has the first RNAsequence at the proximal end, as indicated (indicated with 1 andbrackets). A short hairpin RNA or an extended siRNA can be processed bythe RNAi machinery in the cell to produce an siRNA (3), which can alsobe a double stranded RNA according to the invention, of which one strandcomprising the first RNA sequence can be incorporated into the RISCcomplex (4). A double stranded RNA can be comprised in a pri-miRNAsequence (5) or a pre-miRNA sequence (6). The pri-miRNA can be processedby the RNAi machinery to produce a pre-miRNA and subsequently a maturemiRNA duplex (7), of which one strand comprising the first RNA sequencecan be incorporated into the RISC complex (4). The position of the firstRNA sequence in the pre-miRNA, pri-miRNA and miRNA duplex is indicated 1and brackets. (C) DNA sequence of the pVD-CMV-miH12-155 expressioncassette (CMV promoter (1-588), intervening sequence, Green FluorescentProtein GFP sequence (713-1432), 5′ pri-miRNA flank (1433-1514), 5′pre-miRNA, Guide strand (first RNA sequence) (1520-1540), loop sequence,Passenger strand (second RNA sequence) (1560-1578), 3′ pre-miRNA) 3′pri-miRNA flank (1584-1704), HSV TKpolyA signal (1705-1976); (D) DNAsequence of the pVD-CAG-miH12-451 (CAG promoter (43-1715), 5′ pri-miRNAflank (1716-2017), 5′ pre-miRNA, Guide strand (first RNA sequence)(2034-2054), second RNA sequence &, 3′ pre-miRNA, 3′ pri-miRNA flank(2090-2320), hGH polyA signal (2321-2417) and (E) DNA sequence of thepVD-PGK-miH12-451 expression cassette (PGK promoter (23-277), 5′pri-miRNA flank (278-794), 5′ pre-miRNA, Guide strand (first RNAsequence) (811-831), second RNA sequence &, 3′ pre-miRNA, 3′ pri-miRNAflank (867-1097), hGH polyA signal (1098-1194). (F) pri-miH12-155sequence that is encoded by pVD-CMV-miH12-155. (G) pri-miH12-451sequence that is encoded by pVD-CAG-miH12-451. (G) pri-miH12-451sequence that is encoded by pVD-PGK-miH12-451. For figure (E), (F) and(G), the font type is the same as used above for the corresponding DNA.Promoter sequences are bold, Green Fluorescent protein sequence is initalics underlined (only C), pri-miRNA sequences have a normal fonttype, guide strand (first RNA sequence) is in bold italics and thepassenger strand or second RNA sequence is in italics, pre-miRNAsequences are underlined and the polyA signal is bold underlined.

FIGS. 3A and 3B. In vitro knockdown efficacy of miH1-21. (A) Total HTTknockdown by targeting exon 1. LucHTT was co-transfected in Hek293Tcells with miH1-miH21. Renilla and Firefly luciferase fluorescence wasmeasured 48 h post-transfection. miScr (ctrl) was used as a negativecontrol and was set at 100%. miH12 showed strongest knockdownefficiency.

(B) LucHTT knockdown was by synthetic siH12 with 19-23 nucleotides oflength.

FIGS. 4A and 4B. In vitro knockdown efficacy of miH12-451 with differentpromoters. (A) LucHTT reporter has been co-transfected with CAG-miH12 orPGK-miH12 variants and knockdown efficacy was determined as describedabove for FIG. 3. (B) Passenger (*) strand activity of miH12-451*expressed from the CAG or PGK promoters was measured on specific LucHTT*reporters. No passenger strand activity was detected.

FIG. 5. In vivo efficacy of AAV5-delivered miH12. (A) Experimental setup. Mice were co-injected with AAV5-Luc73QHTT and AAV5-CMV-miScr-155 orAAV5-CMV-miH12-155 in 1:5 ratio. Measurement points are indicated witharrows; (B) AAV5-Luc73QHTT knockdown in animals 6 weeks p.i. wasmeasured by IVIS; (C) AAV5-Luc73QHTT knockdown trend by AAV5-miH12 up to6 weeks p.i.

FIGS. 6A-6D. Human HTT knockdown proof of concept in rat HD mechanisticmodel. (A) Experimental set up; (B) brain histology showing lessneurodegeneration (DARP32) and less mutant Htt (EM48) aggregates in theAAV5-CMV-miH12-155 group; (C) GFP brain histology; (D) Iba1 immuneactivation marker brain histology.

FIGS. 7A and 7B. Human HTT knockdown in the humanized Hu97/18 HD mousemodel. (A) Transduction efficiency in murine brain upon slowintrastriatal injection, convection enhanced diffusion (CED)intrastriatal injection or intracerebral ventricular (ICV) injection ofAAV5-CMV-miH12-155. GFP fluorescence was viewed 5 weeks post injection.(B) Western blot measuring human HTT knockdown in murine brain uponAAV5-miHTT delivery. (C) HTT western blot quantification.

FIGS. 8A-8C. Comparison of selected H12 target with prior art targetsequences. LucHTT was co-transfected in Hek293T cells with the indicatedsiRNAs (A) and miRNA constructs (B and C). Renilla and Fireflyluciferase fluorescence was measured 48 h post-transfection. miH12 andsiH12 showed strongest knockdown efficiency.

DETAILED DESCRIPTION

The present invention provides for a double stranded RNA comprising afirst RNA sequence and a second RNA sequence wherein the first andsecond RNA sequence are substantially complementary, wherein the firstRNA sequence has a sequence length of at least 19 nucleotides and issubstantially complementary to SEQ ID NO. 1.

SEQ ID NO.1 (5′-CUUCGAGUCCCUCAAGUCCUU-3′) corresponds to a targetsequence of the huntingtin gene of exon 1 (SEQ ID NO. 2). Exon 1, asdepicted in FIG. 1B has 21 repeat CAG sequences from nt. 367-429. Theexon 1 sequence as depicted in FIG. 1B corresponds to a normalhuntingtin gene that is not associated with disease. Correspondingmutant huntingtin genes associated with Huntington's disease comprisemuch more than 21 CAG repeat sequences. As said, with 36 to 39 CAGrepeats one may develop signs and symptoms of Huntington's disease,while with 40 or more repeats one almost always develop the disorder.The target sequence SEQ ID NO.1 is comprised in substantially all exon 1sequences, irrespective of the number of CAG repeats.

SEQ ID NO. 1 corresponds to nucleotide nrs. 345-365 of SEQ ID NO.2. 18different target sequences in exon 1 were tested for targeting usingdouble stranded RNAs that were designed to induce a sequence specificinhibition of SEQ ID NO.2. (see FIG. 1 and FIG. 3A) and it was foundthat in particular targeting this sequence from exon 1 was useful inreducing huntingtin gene expression. siRNAs varying in length, i.e.consisting of 19, 20, 21, 22, and 23 consecutive basepairs with 2nucleotide overhangs in addition were found to be effective against thissequence, as well as two separate miRNA scaffolds carrying a 21nucleotide sequence complementary to SEQ ID NO.1 at the guide sequenceposition (see FIGS. 3A, 3B and 4). Hence, the first RNA sequence that issubstantially complementary to the huntingtin target sequence SEQ IDNO.1 has a sequence length of at least 19 nucleotides.

The first RNA sequence according to the invention is comprised in theguide strand of the double stranded RNA, also referred to as antisensestrand as it is complementary (“anti”) to the sense target sequence. Thesecond RNA sequence is comprised in the passenger strand, also referredto as “sense strand” as it may have substantial sequence identity withor be identical with the target sequence. The first and second RNAsequences are comprised in a double stranded RNA and are substantiallycomplementary. The said double stranded RNA according to the inventionis to induce RNA interference to thereby reduce both huntingtin mutantand wild type gene expression. Hence, it is understood thatsubstantially complementary means that it is not required to have allthe nucleotides of the first and second RNA sequences base paired, i.e.to be fully complementary, or all the nucleotides of the first RNAsequence and SEQ ID NO.1 base paired. As long as the double stranded RNAis capable of inducing RNA interference to thereby sequence specificallytarget a sequence comprising SEQ ID NO.1, such substantialcomplementarity is contemplated in the invention.

Hence, in one embodiment the double stranded RNA according to theinvention comprising a first RNA sequence and a second RNA sequencewherein the first and second RNA sequence are substantiallycomplementary, and wherein the first RNA sequence has a sequence lengthof at least 19 nucleotides and is substantially complementary to SEQ IDNO. 1, is capable of inducing RNA interference to sequence specificallyreduce expression of an RNA transcript comprising SEQ ID NO.1. In afurther embodiment, said induction of RNA interference to reduceexpression of an RNA transcript comprising SEQ ID NO.1 means that it isto reduce human Huntingtin gene expression.

One can easily determine whether this is the case by using standardluciferase reporter assays and appropriate controls such as described inthe examples and as known in the art (Zhuang et al. 2006 Methods MolBiol. 2006; 342:181-7). For example, a luciferase reporter comprisingSEQ ID No.1 can be used to show that the double stranded RNA accordingto the invention is capable of sequence specific knock down.Furthermore, as shown in the example section, Huntingtin expression canbe determined with specific antibodies to determine the amount ofexpression in a western blot analysis, as can northern blot analysisdetecting the amount of RNA transcript.

Hence, the double stranded RNA according to the invention is for use ininducing RNA interference. The double stranded RNA according to theinvention is for use in reducing expression of transcripts comprisingSEQ ID NO.1, such as for example SEQ ID NO.2 or the like with varyingnumber of CAG repeats.

As said, the double stranded RNA is capable of inducing RNAinterference. Double stranded RNA structures are well known in the artthat are suitable for inducing RNAi. For example, a small interferingRNA (siRNA) comprises two separate RNA strands, one strand comprisingthe first RNA sequence and the other strand comprising the second RNAsequence. An siRNA design that is often used involves 19 consecutivebase pairs with 3′ two-nucleotide overhangs (see FIG. 2A). This designis based on observed Dicer processing of larger double stranded RNAsthat results in siRNAs having these features. The 3′-overhang may becomprised in the first RNA sequence. The 3′-overhang may be in additionto the first RNA sequence. The length of the two strands of which ansiRNA is composed may be 19, 20, 21, 22, 23, 24, 25, 26 or 27nucleotides or more. Each of the two strands comprises the first andsecond RNA sequence. The strand comprising the first RNA sequence mayalso consist thereof. The strand comprising the first RNA sequence mayalso consist of the first RNA sequence and the overhang sequence.

siRNAs may also serve as Dicer substrates. For example, a Dicersubstrate may be a 27-mer consisting of two strands of RNA that have 27consecutive base pairs. The first RNA sequence is positioned at the3′-end of the 27-mer duplex. At the 3′-end, like the with siRNAs, is atwo nucleotide overhang. The 3′-overhang may be comprised in the firstRNA sequence. The 3′-overhang may be in addition to the first RNAsequence. 5′ from the first RNA sequence, additional sequences may beincluded that are either complementary to the target sequence adjacentto SEQ ID NO.1 or not. The other end of the siRNA dicer substrate isblunt ended. This dicer substrate design results in a preference inprocessing by Dicer such that an siRNA is formed like the siRNA designas described above, having 19 consecutive base pairs and 2 nucleotideoverhangs at both 3′-ends. In any case, siRNAs, or the like, arecomposed of two separate RNA strands (Fire et al. 1998, Nature. 1998Feb. 19; 391(6669):806-11) each RNA strand comprising or consisting ofthe first and second RNA sequence according to the invention.

The double stranded RNA according to the invention does not require bothfirst and second RNA sequences to be comprised in two separate strands.The first and second RNA sequences can also be comprised in a singlestrand of RNA, such as e.g. an shRNA. A shRNA may comprise from5′-second RNA sequence-loop sequence-first RNA sequence-optional 2 ntoverhang sequence-3′. Alternatively, a shRNA may comprise from 5′-firstRNA sequence-loop sequence-second RNA sequence-optional 2 nt overhangsequence-3′. Such an RNA molecule forms intramolecular base pairs viathe substantially complementary first and second RNA sequence. Suitableloop sequences are well known in the art (i.a. as shown in Dallas et al.2012 Nucleic Acids Res. 2012 October; 40(18):9255-71 and Schopman etal., Antiviral Res. 2010 May; 86(2):204-11).

The loop sequence may also be a stem-loop sequence, whereby the doublestranded region of the shRNA is extended. Without being bound by theory,like the siRNA dicer substrate as described above, a shRNA is usuallyprocessed by Dicer to obtain e.g. an siRNA having an siRNA design suchas described above, having e.g. 19 consecutive base pairs and 2nucleotide overhangs at both 3′-ends. In case the double stranded RNA isto be processed by Dicer, it is preferred to have the first and secondRNA sequence at the end of

A double stranded RNA according to the invention may also beincorporated in a pre-miRNA or pri-mi-RNA scaffold. Micro RNAs, i.e.miRNA, are guide strands that originate from double stranded RNAmolecules that are expressed e.g. in mammalian cells. A miRNA isprocessed from a pre-miRNA precursor molecule, similar to the processingof a shRNA or an extended siRNA as described above, by the RNAimachinery and incorporated in an activated RNA-induced silencing complex(RISC) (Tijsterman M, Plasterk R H. Dicers at RISC; the mechanism ofRNAi. Cell. 2004 Apr. 2; 117(1):1-3). Without being bound by theory, apre-miRNA is a hairpin molecule that can be part of a larger RNAmolecule (pri-miRNA), e.g. comprised in an intron, which is firstprocessed by Drosha to form a pre-miRNA hairpin molecule. The pre-miRNAmolecule is a shRNA-like molecule that can subsequently be processed bydicer to result in an siRNA-like double stranded duplex. The miRNA, i.e.the guide strand, that is part of the double stranded RNA duplex issubsequently incorporated in RISC. An RNA molecule such as present innature, i.e. a pri-miRNA, a pre-miRNA or a miRNA duplex, may be used asa scaffold for producing an artificial miRNA that specifically targets agene of choice. Based on the predicted RNA structure, e.g. as predictedusing e.g. m-fold software, the natural miRNA sequence as it is presentin the RNA structure (i.e. duplex, pre-miRNA or pri-miRNA), and thesequence present in the structure that is complementary therewith areremoved and replaced with a first RNA sequence and a second RNA sequenceaccording to the invention. The first RNA sequence and the second RNAsequence may be selected such that the RNA structures that are formed,i.e. pre-miRNA, pri-miRNA and/or miRNA duplex, resemble thecorresponding predicted original sequences. pre-miRNA, pri-miRNA andmiRNA duplexes (that consist of two separate RNA strands that arehybridized via complementary base pairing), as found in nature often arenot fully base paired, i.e. not all nucleotides that correspond with thefirst and second strand as defined above are base paired, and the firstand second strand are often not of the same length. How to use miRNAprecursor molecules as scaffolds for any selected target sequence andsubstantially complementary first RNA sequence is described e.g. in LiuY P Nucleic Acids Res. 2008 May; 36(9):2811-24.

In any case, as is clear from the above, the double stranded RNAcomprising the first and second RNA sequence can comprise additionalnucleotides and/or nucleotide sequences. The double stranded RNA may becomprised in a single RNA sequence or comprised in two separate RNAstrands. Without being bound by theory, whatever design is used for thedouble stranded RNA, it is designed such that an antisense sequencecomprising the first RNA sequence of the invention can be processed bythe RNAi machiney such that it can be incorporated in the RISC complexto have its action. The said sequence comprising or consisting of thefirst RNA sequence of the invention being capable of sequencespecifically targeting SEQ ID NO.1. Hence, as long as the doublestranded RNA is capable of inducing RNAi, such a double stranded RNA iscontemplated in the invention. Hence, in one embodiment, the doublestranded RNA according to the invention is comprised in a pre-miRNAscaffold, a pri-miRNA scaffold, a shRNA, or an siRNA.

The term complementary is defined herein as nucleotides of a nucleicacid sequence that can bind to another nucleic acid sequence throughhydrogen bonds, i.e. nucleotides that are capable of base pairing.Ribonucleotides, the building blocks of RNA are composed of monomers(nucleotides) containing a sugar, phosphate and a base that is either apurine (guanine, adenine) or pyrimidine (uracil, cytosine).Complementary RNA strands form double stranded RNA. A double strandedRNA may be formed from two separate complementary RNA strands or the twocomplementary RNA strands may be comprised in one RNA strand. Incomplementary RNA strands, the nucleotides cytosine and guanine (C andG) can form a base pair, guanine and uracil (G and U), and uracil andadenine (U and A). The term substantial complementarity means that isnot required to have the first and second RNA sequence to be fullycomplementary, or to have the first RNA sequence and SEQ ID NO.1 to befully complementary. For example, the first and second nucleotides asshown in FIG. 2A are substantially complementary and not fullycomplementary.

In one embodiment, the substantial complementarity between the first RNAsequence and SEQ ID NO.1 consists of having no mismatches, onemismatched nucleotide, or two mismatched nucleotides. It is understoodthat one mismatched nucleotide means that over the entire length of thefirst RNA sequence that base pairs with SEQ ID NO.1 one nucleotide doesnot base pair with SEQ ID NO.1. Having no mismatches means that allnucleotides base pair with SEQ ID NO.1, and having 2 mismatches meanstwo nucleotides do not base pair with SEQ ID NO.1. The first RNAsequence may also be longer than 21 nucleotides, in this scenario, thesubstantial complementarity is determined over the entire length of SEQID NO.1. This means that SEQ ID NO.1 in this embodiment has either no,one or two mismatches over its entire length when base paired with thefirst RNA sequence. For example, as shown in FIG. 3B and the examples,siRNAs having a first nucleotide sequence length of 22 and 23nucleotides were tested. These first nucleotide sequences had nomismatches and were fully complementary to SEQ ID NO.1. Having a fewmismatches between the first nucleotide sequence and SEQ ID NO.1 may beallowed according to the invention, as long as the double stranded RNAaccording to the invention is capable of reducing expression oftranscripts comprising SEQ ID NO.1, such as a luciferase reporter ore.g. a transcript comprising SEQ ID NO.1. In this embodiment,substantial complementarity between the first RNA sequence and SEQ IDNO.1 consists of having no, one or two mismatches over the entire lengthof either the first RNA sequence or SEQ ID NO.1, whichever is theshortest.

In one embodiment the first RNA sequence and SEQ ID NO.1 have at least15, 16, 17, 18, or 19 nucleotides that base pair. Preferably the firstRNA sequence and SEQ ID NO. 1 are substantially complementary, saidcomplementarity comprising at least 19 base pairs. In anotherembodiment, the first RNA sequence has at least 8, 9, 10, 11, 12, 13 or14 consecutive nucleotides that base pair with consecutive nucleotidesof SEQ ID NO.1. In another embodiment, the first RNA sequence has atleast 19 consecutive nucleotides that base pair with consecutivenucleotides of SEQ ID NO.1. In another embodiment the first RNA sequencecomprises at least 19 consecutive nucleotides that base pair with 19consecutive nucleotides of SEQ ID NO.1. In still another embodiment, thefirst RNA sequence has at least 17 nucleotides that base pair with SEQID NO.1 and has at least 15 consecutive nucleotides that base pair withconsecutive nucleotides of SEQ ID NO.1. The sequence length of the firstnucleotide is at most 21, 22, 23, 24, 25, 26, or 27 nucleotides.

As said, a mismatch according to the invention means that a nucleotideof the first RNA sequence does not base pair with SEQ ID NO.1.Nucleotides that do not base pair are A and C, C and U, or A and G. Amismatch may also result from a deletion of a nucleotide, or aninsertion of a nucleotide. When the mismatch is a deletion in the firstRNA sequence, this means that a nucleotide of SEQ ID NO.1 is not basepaired with the first RNA sequence when compared with the entire lengthof the first RNA sequence. Nucleotides that can base pair are A-U, G-Cand G-U. A G-U base pair is also referred to as a G-U wobble, or wobblebase pair. In one embodiment the number of G-U base pairs between thefirst RNA sequence and SEQ ID NO.1 is 0, 1 or 2. In one embodiment,there are no mismatches between the first RNA sequence and SEQ ID NO.1and a G-U base pair or G-U pairs are allowed. Preferably, there may beno G-U base pairs between the first RNA sequence and SEQ ID NO.1, or thefirst RNA sequence and SEQ ID NO.1 only have base pairs that are A-U orG-C. Preferably, there are no G-U base pairs and no mismatches betweenthe first RNA sequence and SEQ ID NO.1. Hence, the first RNA sequence ofthe double stranded RNA according to invention preferably is fullycomplementary to SEQ ID NO.1, said complementarity consisting of G-C andA-U base pairs.

It may be not required to have full complementarity (i.e. full basepairing (no mismatches) and no G-U base pairs) between the firstnucleotide sequence and SEQ ID NO.1 as such a first nucleotide sequencecan still allow for sufficient suppression of gene expression. Also,having not full complementarity may be contemplated for example to avoidor reduce off-target sequence specific gene suppression whilemaintaining sequence specific inhibition of transcripts comprising SEQID NO.1. However, it may be preferred to have full complementarity as itmay result in more potent inhibition. Without being bound by theory,having full complementarity between the first RNA sequence and SEQ IDNO.1 may allow for the activated RISC complex comprising the said firstRNA sequence to cleave its target sequence, whereas having mismatchesmay only allow inhibition of translation, the latter resulting in lesspotent inhibition.

In one embodiment, the first RNA sequence has a sequence length of atleast 19 nucleotides, preferably 20 nucleotides, more preferably of atleast 21 nucleotides. The sequence length can also be at least 22nucleotides, or at least 23 nucleotides. The first RNA sequenceaccording to the invention may be selected from SEQ ID NOs. 3-7.

TABLE 1 First RNA sequences. SEQ ID NO. First RNA sequence length 35′-AAGGACUUGAGGGACUCGA-3′ 19 4 5′-AAGGACUUGAGGGACUCGAA-3′ 20 55′-AAGGACUUGAGGGACUCGAAG-3′ 21 6 5′-AAGGACUUGAGGGACUCGAAGG-3′ 22 75′-AAGGACUUGAGGGACUCGAAGGC-3′ 23

The first RNA sequences of table 1 have been shown to specificallyinhibit transcripts comprising SEQ ID NO.1 as described in the examplesection.

In one embodiment, the first nucleotide sequence of the double strandedRNA according to the invention are fully complementary with SEQ ID NO.1.This means that there are no mismatches between the first RNA sequenceand SEQ ID NO.1 over the entire length of either the first RNA sequenceor SEQ ID NO.1, whichever is the shortest. Preferably, the firstnucleotide sequence and SEQ ID NO.1 are fully complementary, comprisingonly G-C and A-U base pairs. Preferably, the first RNA sequence isselected from the group consisting of SEQ ID NOs 3-7, which are fullycomplementary with SEQ ID NO.1. Most preferably the first RNA sequenceis SEQ ID NO. 5. When the first nucleotide sequence is 21 nucleotides orless (SEQ ID NOs. 3, 4 and 5) all nucleotides of the first nucleotidesequence base pair with SEQ ID NO.1. When the first nucleotide sequenceis longer than 21 nucleotides (SEQ ID NOs. 6 and 7), all nucleotides ofSEQ ID NO.1 base pair with the first nucleotide sequence. The additionalnucleotides that are comprised in the first RNA sequence do not basepair with SEQ ID NO.1. When the first nucleotide sequence is longer than21 nucleotides and the additional nucleotides are to be part of theguide strand, preferably the additional nucleotides are complementary tothe sequence flanking sequence of SEQ ID NO.1 as present in SEQ ID NO.2.

With regard to the second RNA sequence, the second RNA sequence issubstantially complementary with the first RNA sequence. The second RNAsequence combined with the first RNA sequence forms a double strandedRNA. As said, this is to form a suitable substrate for the RNAinterference machinery such that a guide sequence derived from the firstRNA sequence is comprised in the RISC complex in order to sequencespecifically inhibit expression of its target, i.e. Huntingtin geneexpression. As said, such double stranded RNA is preferably comprised ina pre-miRNA scaffold, a pri-miRNA scaffold, a shRNA, or an siRNA.

The sequence of the second RNA sequence has similarities with the targetsequence. However, the substantial complementarity with the first RNAsequence may be selected to have less substantial complementarity ascompared with the substantial complementarity between the first RNAsequence and SEQ ID NO.1. Hence, the second RNA sequence may comprise 0,1, 2, 3, 4, or more mismatches, 0, 1, 2, 3, or more G-U wobble basepairs, and may comprise insertions of 0, 1, 2, 3, 4, nucleotides and/ordeletions of 0, 1, 2, 3, 4, nucleotides. Preferably the first RNAsequence and the second RNA sequence are substantially complementary,said complementarity comprising 0, 1, 2 or 3 G-U base pairs and/orwherein said complementarity comprises at least 17 base pairs.

These mismatches, G-U wobble base pairs, insertions and deletions, arewith regard to the first RNA sequence, i.e. the double stranded regionthat is formed between the first and second RNA sequence. As long as thefirst and second RNA sequence can substantially base pair, and arecapable of inducing sequence specific inhibition of SEQ ID NO.1, suchsubstantial complementarity is allowed according to the invention. It isalso understood that substantially complementarity between the first RNAsequence and the second RNA sequence may depend on the double strandedRNA design of choice. It may depend for example on the miRNA scaffoldthat is chosen for in which the double stranded RNA is to beincorporated.

TABLE 2 Second RNA sequences. SEQ ID NO. Second RNA sequence length  85′-UCGAGUCCCUCAAGUCCUU-3′ 19  9 5′-UUCGAGUCCCUCAAGUCCUU-3′ 20 105′-CUUCGAGUCCCUCAAGUCCUU-3′ 21 11 5′-CCUUCGAGUCCCUCAAGUCCUU-3′ 22 125′-GCCUUCGAGUCCCUCAAGUCCUU-3′ 23 13 5′-CUUCGAGUCUCAAGUCCUU-3′ 19 145′-ACGAGUCCCUCAAGUCCUC-3′ 19

In one embodiment, a second RNA sequence is selected from the groupconsisting of SEQ ID NOs. 8-14. In Table 2, examples of said second RNAsequences in accordance with the invention are listed. SEQ ID NOs. 8, 9,10, 11 and 12 are fully complementary with SEQ ID NO.1 over their entirelength. SEQ ID NOs. 13 and 14 can be combined with a first nucleotidehaving a sequence corresponding to SEQ ID NO.5 of 21 nucleotides, whichis complementary with SEQ ID NO.1 over its entire length. SEQ ID NO.13is complementary with SEQ ID NO.5 having a two nucleotide deletion(resulting in the corresponding 2 nucleotides of SEQ ID NO.5 not basepaired) and 19 nucleotides base paired. SEQ ID NO.14 is complementarywith SEQ ID NO.5 having a two nucleotides deletion, two mismatches, and17 nucleotides base paired. The complementarity can also be seen in FIG.2B, as the combination of SEQ ID NO.5 and SEQ ID NO.13 is present inmiH12_155, and the combination of SEQ ID NO.5 and SEQ ID NO.14 ispresent in miH12_451a. Hence, as is clear from the above, the second RNAsequence does not require complementarity with the first RNA sequence,but may comprise deletions, insertions and mutations that result inmismatches, as compared with SEQ ID NO.1.

As is clear from the above, the substantial complementarity between thefirst RNA sequence and the second RNA sequence, may comprise mismatches,deletions and/or insertions relative to a first and second RNA sequencebeing fully complementary (i.e. fully base paired). In one embodiment,the first and second RNA sequences have at least 11 consecutive basepairs. Hence, at least 11 consecutive nucleotides of the first RNAsequence and at least 11 consecutive nucleotides of the second RNAsequence are fully complementary. In another embodiment the first andsecond RNA sequence have at least 15 nucleotides that base pair. Saidbase pairing between at least 15 nucleotides of the first RNA sequenceand at least 15 nucleotides of the second RNA sequence may consist ofG-U, G-C and A-U base pairs, or may consist of G-C and A-U base pairs.In still another embodiment, the first and second RNA sequence have atleast 15 nucleotides that base pair and have at least 11 consecutivebase pairs. In still another embodiment, the first RNA sequence and thesecond RNA sequence are substantially complementary, wherein saidcomplementarity comprises at least 17 base pairs. Said 17 base pairs maypreferably be 17 consecutive base pairs, said base pairing consisting ofG-U, G-C and A-U base pairs or consisting of G-C and A-U base pairs.

In one embodiment, the first and second nucleotide sequence are selectedfrom the group of SEQ ID NOs. 3 and 8; 4 and 9; 5 and 10; 5 and 13; 5and 14; 6 and 11; and 7 and 12. These combinations of first and secondnucleotide sequences were shown to be effective when comprised in siRNAsor miRNA scaffolds.

The first and second nucleotide sequences that are substantiallycomplementary preferably do not form a double stranded RNA of 30consecutive base pairs or longer, as these can trigger an innate immuneresponse via the double-stranded RNA (dsRNA)-activated protein kinasepathway. Hence, the double stranded RNA is preferably less than 30consecutive base pairs. Preferably, a pre-miRNA scaffold, a pri-miRNAscaffold, a shRNA, or an siRNA comprising the double stranded RNAaccording to the invention does not comprise 30 consecutive base pairs.

Preferably the double stranded RNA according to the invention iscomprised in a pre-miRNA or pri-miRNA scaffold. A pri-miRNA scaffoldcomprises a pre-miRNA scaffold. The pre-miRNA scaffold comprises thedouble stranded RNA of the invention, i.e. the first RNA sequence andthe second RNA sequence. Preferably, the double stranded DNA accordingto the invention is comprised in a pri-miRNA scaffold derived frommiR-451a (also referred to as miR-451) or miR-155. Examples of doublestranded RNAs according to the invention comprised in a pre-miRNAscaffold are depicted in FIG. 2A. The sequence of these pre-miRNAs arelisted in table 3 below.

TABLE 3 Pre-miRNA scaffolds with SEQ ID NO. 5 SEQ ID NO. Name Sequence15 pre- 5′-CUUGGGAAUGGCAAGGAAGGACU miR451a UGAGGGACUCGAAGACGAGUCCCUCAAGUCCUCUCUUGCUAUACCCAGA-3′ 16 pre- 5′-UGCUGAAGGACUUGAGGGACUCG miR155AAGGUUUUGGCCACUGACUGACCUUC GAGUCUCAAGUCCUUCAGGA-3′

The pre-mRNA sequence of SEQ ID NO.15 consists of a 5′ arm correspondingto nucleotides 1-16 of SEQ ID NO.15, a first RNA sequence correspondingto SEQ ID NO.5 from nucleotides 17-37 of SEQ ID NO.15, a second RNAsequence corresponding to SEQ ID NO.14 from nucleotides 38-56 of SEQ IDNO.15, and a 3′-arm corresponding to nucleotides 57-72 of SEQ ID NO.15.The pre-miR451a scaffold according to the invention comprises the 5′-armcorresponding to nucleotides 1-16 of SEQ ID NO.15, followed by the firstnucleotide sequence according to the invention, the second nucleotidesequence according to the invention, and the 3′-arm corresponding tonucleotides 57-72 of SEQ ID NO.15. Preferably, the first and second RNAsequences are selected such that when comprised in the pri-miR451ascaffold a predicted structure highly similar or as shown in FIG. 2B isobtained. Preferably the base pairs that are formed between the firstand second RNA sequence are G-C, G-U and A-U base pairs and preferablythe sequence length of the first RNA sequence is 21 nucleotides and thelength of the second RNA sequence is preferably 19 nucleotides.Preferably the base pairs that are formed between the first and secondRNA sequence are G-C and A-U base pairs and preferably the sequencelength of the first RNA sequence is 21 nucleotides and the length of thesecond RNA sequence is preferably 19 nucleotides. Without being bound bytheory, this pri-R451a scaffold may be preferred as it does not resultin a passenger strand to be processed by the RNAi machinery to beincorporated into RISC (Cheloufi et al., 2010 Jun. 3; 465(7298):584-9).From an siRNA or miRNA duplex, in principle both strands can beincorporated into RISC. As the passenger strand (corresponding to thesecond sequence) may result in targeting of transcripts other than ahuntingtin transcript, using the pri-miR451a or pre-miR451a scaffold mayallow one to avoid such unwanted targeting. When tested for potential“passenger strand” activity, no activity was detected with a pri-451ascaffold (see FIGS. 4A and 4B). The processing of the pre-miRNA hairpinis understood to be Dicer independent and to be cleaved by Ago2 (Yang etal., Proc Natl Acad Sci USA. 2010 Aug. 24; 107(34):15163-8).

The pre-mRNA sequence of SEQ ID NO.16 consists of a 5′ arm correspondingto nucleotides 1-5 of SEQ ID NO.16, a first RNA sequence correspondingto SEQ ID NO.5 from nucleotides 6-26 of SEQ ID NO.16, a loop sequencefrom nucleotides 27-45 of SEQ ID NO.16, a second RNA sequencecorresponding to SEQ ID NO.13 from nucleotides 46-64 of SEQ ID NO.16,and a 3′-arm corresponding to nucleotides 65-69 of SEQ ID NO.16. Thepre-155 scaffold comprising the first and second RNA sequence accordingto the invention comprises the 5′-arm corresponding to nucleotides 1-5of SEQ ID NO.16, the first RNA sequence, a loop sequence correspondingto nucleotides 27-45 of SEQ ID NO.16, the second RNA sequence, and a3′-arm corresponding to nucleotides 65-69 of SEQ ID NO.16. Preferably,the first and second RNA sequence are selected such that when comprisedin the pre-miR155 scaffold (or pri-miRNA scaffold) a predicted structurehighly similar as shown in FIG. 2B is obtained. Preferably the basepairs that are formed between the first and second RNA sequence are G-Cor A-U base pairs and preferably the sequence length of the first RNAsequence is 21 nucleotides and the length of the second RNA sequence ispreferably 19 nucleotides. Said 19 nucleotides are preferably fullycomplementary with nucleotides 2-18 of a first nucleotide sequence of 21nucleotides in length.

A pre-miRNA sequence when comprised in a larger RNA sequence requires5′- and 3′-single stranded flanking sequences that allow Drosharecognition and cleavage. Said sequences that are suitable to allow forDrosha recognition and cleavage are 5′-pri-miRNA sequence and the3′-pri-miRNA sequence. For example, the expressed pre-miH12_155 sequenceas depicted in FIG. 5A is flanked by 5′-pri-miRNA and the 3′-pri-miRNAsequences of miR155 in the expression vector CMV-miH12-155 (FIG. 2C).The pri-miRNA sequence comprising the miRH12_155 is listed in FIG. 2F,the 5′-pri-miRNA corresponding to nucleotides 1-87 and the 3′-pri-miRNAcorresponding to 147-272. Likewise, the CAG-miH12-451 and PGK-miH12-451pri-miRNA sequences that are expressed by their respective vectors inFIGS. 2D and 2E are shown in FIGS. 2G and 2H. The 5′-pri-miRNA and the3′-pri-miRNA of the expressed CAG-miH12-451 RNA corresponding tonucleotides 1-302 and 375-605, and the 5′-pri-miRNA and the 3′-pri-miRNAof the expressed of PGK-miH12-451 RNA corresponding respectively tonucleotides 1-516 and 589-819. The length of the single-stranded flankscan vary but is typically around 80 nt (Zeng and Cullen, J Biol Chem.2005 Jul. 29; 280(30):27595-603; Cullen, Mol Cell. 2004 Dec. 22;16(6):861-5) The minimal length of the single-stranded flanks can easilybe determined as when it becomes too short, Drosha processing may failand sequence specific inhibition will be reduced or even absent. In oneembodiment, the pri-miRNA scaffold carrying the first and second RNAsequence according to the invention has a 5′-sequence flank and a 3′sequence flank relative to the predicted remiRNA structure of at least50 nucleotides. The pre-miRNA and the pri-miRNA derived sequences arepreferably all derived from the same naturally occurring pri-miRNAsequence.

The pre-miRNA sequence of SEQ ID NO.15 and SEQ ID NO.16 are encoded bythe DNA sequences as depicted in FIGS. 2C (SEQ ID NO.16), 2D (SEQ IDNO.15), and 2E (SEQ ID NO.15). Pri-miRNA sequences comprising saidpre-miRNA sequences are depicted in FIGS. 2F (SEQ ID NO.16), G (SEQ IDNO.15) and H (SEQ ID NO.15). The pri-miRNA encoded by CMV-miH12-155correspond to nucleotides 1433-1704 of FIG. 2C, of CAG-miH12-451 tonucleotides 1716-2320 of FIG. 2D and of PGK-miH12-451 to nucleotides278-1097 of FIG. 2E. Likewise, the first and second RNA sequences are tobe incorporated as described above for the pre-miRNAs.

The double stranded RNAs according to the invention, incorporated in ansiRNA, shRNA, pri-mRNA scaffold or pre-miRNA scaffold can be provided ina cell using methods known in the art, such as lipofection, transfectionor using any other suitable means therefor. The double stranded RNAsaccording to the invention may be synthetic double stranded RNAs ornatural double stranded RNAs. Synthetic double stranded RNAs and maycomprise nucleic acids containing known analogs of natural nucleotides.The said double stranded RNA has similar properties as compared to theirnatural counterparts and an RNA interference activity similar to orimproved over double stranded RNAs that consist entirely ofnon-synthetic double stranded RNA. For example, synthetic siRNAs mayinclude in their design the use of Locked Nucleic Acid (a ribose ringconnected by a methylene bridge (orange) between the 2′-0 and 4′-Catoms), modified nucleotides such as nucleotides comprisingphosphorothioates, 2′-O-Me, 2′-O-allyl and 2′-deoxy-fluorouridine. It iswell known that double stranded RNAs, e.g. siRNAs, can accommodate quitea number of modifications at both base-paired and non-base-pairedpositions without significant loss of activity. Preferably, the doublestranded RNA of the invention is a double stranded RNA that consists ofnatural nucleotides, such as obtained from expression of a doublestranded RNA from.

Hence, in one embodiment, the said double stranded RNAs of the inventionare encoded by a DNA sequence. The said DNA sequence encoding the saiddouble stranded RNA, e.g. as comprised in an siRNA, shRNA, pri-mRNAscaffold or pre-miRNA scaffold, is comprised in an expression cassette.It is understood that when the double stranded RNA is to be e.g. ansiRNA, consisting of two RNA strands, that there are two expressioncassettes required. One encoding an RNA strand comprising the first RNAsequence, the other cassette encoding an RNA strand comprising the firstRNA strand. When the double stranded RNA is comprised in a single RNAmolecule, e.g. encoding a shRNA, pre-miRNA or pri-miRNA, one expressioncassette may suffice. A pol II expression cassette may comprise apromoter sequence a sequence encoding the RNA to be expressed followedby a polyadenylation sequence. In case the double stranded RNA that isexpressed comprises a pri-miRNA scaffold, the encoded RNA sequence mayencode for intron sequences and exon sequences and 3′-UTR's and 3′-UTRs.A pol III expression cassette in general comprises a promoter sequence,followed by the DNA sequence encoding the RNA (e.g. shRNA sequence,pre-miRNA, or a strand of the double stranded RNAs to be comprised ine.g. an siRNA or extended siRNA). A pol I expression cassette maycomprise a pol I promoter, followed by the RNA encoding sequence and a3′-Box. Expression cassettes for double stranded RNAs are well known inthe art, and any type of expression cassette can suffice, e.g. one mayuse a pol III promoter, a pol II promoter or a pol I promoter (i.a. terBrake et al., Mol Ther. 2008 March; 16(3):557-64, Maczuga et al., BMCBiotechnol. 2012 Jul. 24; 12:42). Examples of expression cassettesexpressing a double stranded RNA according to the invention are depictedin FIGS. 2C-E.

Preferably a pol II promoter is used, such as the PGK promoter, a CBApromoter or a CMV promoter (see FIGS. 2C-D). As Huntington's diseaseaffects neurons, it may in particularly be useful to use a neurospecificpromoter. Examples of suitable neurospecific promoters areNeuron-Specific Enolase (NSE), human synapsin 1, caMK kinase andtubuline. Other suitable promoters that can be contemplated areinducible promoters, i.e. a promoter that initiates transcription onlywhen the host cell is exposed to some particular stimulus.

Said expression cassettes according to the invention can be transferredto a cell, using e.g. transfection methods. Any suitable means maysuffice to transfer an expression cassette according to the invention.Preferably, gene therapy vectors are used that stably transfer theexpression cassette to the cells such that stable expression of thedouble stranded RNAs that induce sequence specific inhibition of thehuntingtin gene as described above can be achieved. Suitable vectors maybe lentiviral vectors, retrotransposon based vector systems, or AAVvectors. It is understood that as e.g. lentiviral vectors carry an RNAgenome, the RNA genome will encode for the said expression cassette suchthat after transduction of a cell, the said DNA sequence and saidexpression cassette is formed. Preferably a viral vector is used such asAAV. Preferably the AAV vector that is used is an AAV vector of serotype5. AAV of serotype 5 may be in particularly useful for transducingneurons as shown in the examples. The production of AAV vectorscomprising any expression cassette of interest is well described in;WO2007/046703, WO2007/148971, WO2009/014445, WO2009/104964,WO2011/122950, WO2013/036118, which are incorporated herein in itsentirety.

AAV sequences that may be used in the present invention for theproduction of AAV vectors, e.g. produced in insect or mammalian celllines, can be derived from the genome of any AAV serotype. Generally,the AAV serotypes have genomic sequences of significant homology at theamino acid and the nucleic acid levels, provide an identical set ofgenetic functions, produce virions which are essentially physically andfunctionally equivalent, and replicate and assemble by practicallyidentical mechanisms. For the genomic sequence of the various AAVserotypes and an overview of the genomic similarities see e.g. GenBankAccession number U89790; GenBank Accession number J01901; GenBankAccession number AF043303; GenBank Accession number AF085716; Chloriniet al. (1997, J. Vir. 71: 6823-33); Srivastava et al. (1983, J. Vir.45:555-64); Chlorini et al. (1999, J. Vir. 73:1309-1319); Rutledge etal. (1998, J. Vir. 72:309-319); and Wu et al. (2000, J. Vir. 74:8635-47). AAV serotypes 1, 2, 3, 4 and 5 are preferred source of AAVnucleotide sequences for use in the context of the present invention.Preferably the AAV ITR sequences for use in the context of the presentinvention are derived from AAV1, AAV2, and/or AAV5. Likewise, the Rep52,Rep40, Rep78 and/or Rep68 coding sequences are preferably derived fromAAV1, AAV2 and AAV5. The sequences coding for the VP1, VP2, and VP3capsid proteins for use in the context of the present invention mayhowever be taken from any of the known 42 serotypes, more preferablyfrom AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 or newlydeveloped AAV-like particles obtained by e.g. capsid shufflingtechniques and AAV capsid libraries.

In another embodiment, a host cell is provided comprising the said DNAsequence, or said expression cassette according to the invention. Forexample, the said expression cassette or DNA sequence may be comprisedin a plasmid contained in bacteria. Said expression cassette or DNAsequence may also be comprised in a production cell that produces e.g. aviral vector.

As shown in the example section, and as explained above, the doublestranded RNA according to the invention, the DNA sequence according toinvention, the expression cassette according to the invention and thegene therapy vector according to the invention are for use in a medicaltreatment, in particular for use in the treatment of Huntington'sdisease. Said medical treatment when using an AAV vector (or likewisefor a gene therapy vector) comprising a direct infusion of AAV vector ofthe invention into the brain. Said direct infusion in furtherembodiments comprising an intrathecal infusion of the vector into thecerebrospinal fluid. Such an intrathecal infusion represents anefficient way to deliver the gene therapy vector to the CNS and totarget the neurons. Preferably striatal and cortical structures aretargeted via intrastriatal convection enhanced diffusion (CED) deliveryof AAV vectors through injections into the striatum. More preferably,for a larger coverage of the CNS, injections are into the striatum andinto the thalamus as well. Hence, AAV vectors are deliveredintrastriatally, or delivered intrastriatally and intrathalamicallythrough convection enhanced diffusion (CED) injections in the striatum,or the striatum and the thalamamus. Such injections are preferablycarried out through MRI-guided injections. Said methods of treatmentsare in particular useful for human subjects having Huntington's disease.It is understood that the treatment of Huntington's disease involveshuman subjects having Huntington's disease including human subjectshaving a genetic predisposition of developing Huntington's disease thatdo not yet show signs of the disease. Hence, the treatment of humansubjects with Huntington's disease includes the treatment of any humansubject carrying an Huntingtin allele with more than 35 CAG repeats.

EMBODIMENTS

-   -   1. A double stranded RNA comprising a first RNA sequence and a        second RNA sequence wherein the first and second RNA sequence        are substantially complementary, wherein the first RNA sequence        has a sequence length of at least 19 nucleotides and is        substantially complementarity to SEQ ID NO. 1.    -   2. A double stranded RNA according to embodiment 1, wherein said        double stranded RNA is capable of reducing huntingtin gene        expression.    -   3. A double stranded RNA according to embodiment 1 or embodiment        2, wherein the double stranded RNA is comprised in a pre-miRNA        scaffold, a pri-miRNA scaffold, a shRNA, or an siRNA, preferably        a pre-miRNA scaffold.    -   4. A double stranded RNA according to any one of embodiments        1-3, wherein the first RNA sequence has a sequence length of at        least 20 nucleotides, preferably of at least 21 nucleotides.    -   5. A double stranded RNA according to any one of embodiments        1-4, wherein the first RNA sequence is fully complementary to        SEQ ID. NO.1.    -   6. A double stranded RNA according to any one of embodiments 1-5        wherein the first strand is selected from the group consisting        of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ        ID NO.7.    -   7. A double stranded RNA according to any one of embodiments        1-6, wherein the first strand and second strand are selected        from the group consisting of the combinations of SEQ ID NO. 3        and 8; SEQ ID NO. 4 and 9; SEQ ID NO. 5 and 10; SEQ ID NO. 5 and        13; SEQ ID NO. 5 and 14; SEQ ID NO. 6 and 11; and SEQ ID NO. and        7 and 12.    -   8. A double stranded RNA according to any one of embodiments        1-7, wherein the double stranded RNA is comprised in a pre-miRNA        scaffold derived from miR-451a or miR-155.    -   9. A DNA sequence encoding the double stranded RNA according to        any one of embodiments 1-8.    -   10. An expression cassette encoding a double stranded RNA in        accordance with any one of embodiments 1-9.    -   11. An expression cassette according to embodiment 10, wherein        the expression cassette comprises the PGK promoter, a CMV        promoter, a neurospecific promoter or a CBA promoter.    -   12. A gene therapy vector comprising the expression cassette        according to embodiment 10 or 11.    -   13. A gene therapy vector according to embodiment 12, wherein        the gene therapy vector is an AAV vector, preferably an AAV        vector of serotype 5.    -   14. A host cell comprising the DNA sequence according to        embodiment 9 or the expression cassette according to embodiment        10 or embodiment 11.    -   15. A double stranded RNA according to any one of embodiments        1-8, a DNA sequence according to embodiment 9, an expression        cassette according to embodiment 10 or embodiment 11, a gene        therapy vector according to embodiment 12 or embodiment 13, for        use in a medical treatment.    -   16. A double stranded RNA according to any one of embodiments        1-8, a DNA sequence according to embodiment 9, an expression        cassette according to embodiment 10 or embodiment 11, a gene        therapy vector according to embodiment 12 or embodiment 13, for        use in the treatment of Huntington's disease.

Examples

miRNA Scaffold Expression Constructs & siRNAs

To create miRNA scaffold vectors based on miR155, 21 nucleotide (bp)sequences fully complementary with the selected target sequences in HTTas indicated in FIG. 1, were embedded into the pri-mir-155 backbone ofpcDNA6.2-GW/EmGFP-miR (Invitrogen, Carlsbad, Calif.) resulting inpVD-CMV-miHTT-155 (an example of an expression cassette sequence isdepicted in FIG. 2C, the pre-miRNA and pri-miRNA sequence as comprisedin the expressed RNA are depicted in FIGS. 2B and 2F, respectively). Thepri-mir-155 constructs were designed based on the instructions providedby Invitrogen (BLOCK-iT, Pol II miR RNAi expression Vector Kits, VersonE, Jun. 22, 2007, 25-0857) by annealing synthetic double-strandedoligonucleotides in the BsaI site of pcDNA6.2-GW/emGFP-mir155. Thestructure of all artificial pre-miRNA as encoded by the miHtt constructswas verified using the Mfold software (Nucleic Acids Res. 31 (13),3406-15, (2003), using mfold version 3.5, as available online onhttp://mfold.rna.albany.edu/?q=mfold). The predicted structure of thepre-miRNA-155 scaffold carrying the sequence corresponding with SEQ IDNO.5 is shown in FIG. 2B. The DNA sequence encoding the expressionconstruct for the pri-miRNA-155 scaffold with SEQ ID NO.5 as a selectedfirst sequence is listed in FIG. 2C (SEQ ID NO. 17), this construct isalso referred to as miH12 and its target H12. For the other 20 selectedtarget sequences, the sequences were designed to be fully complementarywith the target sequences as depicted in FIG. 1, and the miRNA scaffoldhaving the same structural features as depicted in FIG. 2B, i.e. thesecond RNA sequence embedded in the scaffold corresponds to the sequenceto which the first RNA sequence selected is fully complementary, buthaving a 2 nucleotide deletion in the center. As a control similarly, ascrambled RNA sequence was used as a first RNA sequence, and like above,a second RNA sequence was designed to create a vector pVD-CMV-miScr-155.The constructs contained GFP for allowing both miRNA expression andtransduction visualization in vitro and in vivo.

A miRNA scaffold vector based on miR451a was created. The DNA sequenceencoding the pri-miR-451 scaffold was synthesized based on the predictedmature mir-451 sequence being replaced by the H12 targeting sequence,i.e. SEQ ID NO.5 as first RNA sequence. The second RNA sequence wasdesigned to be fully complementary to nucleotides 2-18 of the first RNAsequence. The second RNA sequence was selected such that the predictedRNA structure of the artificial pre-miRNA sequence adopted a similarstructure as the original wild-type structure. The structure of thepre-miRNA as encoded by the constructs was verified using the Mfoldsoftware (Nucleic Acids Res. 31 (13), 3406-15, (2003), using mfoldversion 3.5, as available online onhttp://mfold.rna.albany.edu/?q=mfold). The predicted structure using SEQID NO.5 is shown in FIG. 2B. Two different miR451a scaffolds expressingvectors were made. The DNA sequences of the expression constructs aredepicted in FIGS. 2D and 2E, the corresponding respective pri-miRNAscaffold sequences as comprised in the expressed RNA are listed in FIGS.2G and 2H. FIG. 2D shows the DNA sequence of the expression cassette ofpVD-CAG-miH12-451, which expresses the miRNA scaffold with a CAGpromoter, and in FIG. 2E the DNA sequence of the expression cassette ofpVD-PGK-miH12-451 is shown, which uses a PGK promoter.

Synthetic siRNA targeting HTT at the miH12 target, i.e. SEQ ID NO.1,were designed with lengths of 19-23 bp (Table 1). The siRNAs comprisedfirst and second nucleotide sequence corresponding to SEQ ID NOs. 3 and8; 4 and 9; 5 and 10; 6 and 11; and 7 and 12. The siRNAs were designedto have 3′-UU overhangs in both strands.

Reporter Constructs

The psiCheck-2 constructs LucHTT containing the complete HTT exon 1sequence was designed and cloned following the instructions as providedby Promega (siCHECK™ Vectors, C8011, Promega Benelux b.v., Leiden, TheNetherlands). LucHTT comprises SEQ ID NO.1 and flanking sequencesthereof as present in SEQ ID NO.2 (see FIG. 1B). The LucH12_451areporter comprises the sequence complementary to the second RNA sequenceas designed to be expressed by pVD-CAG-miH12-451 and pVD-PGK-miH12-451.All constructs have been sequenced, and the correct sequence has beenverified. The knockdown efficacy of all miRNA scaffolds and siRNAs weredetermined on specific luciferase reporters in vitro. Hek293T cells wereco-transfected with the miHtt and the Luciferase reporter in a 1:1 ratio(miR-155, PGK-miH12-451), or 1:10 ratio (CAG-miH12-451, i.e. the CAGpromoter is very strong). Renilla luciferase knockdown was measured 48 hpost transfection (p.t.), and Firefly was measured as an internalcontrol. miScr was used as a negative control and was set at 100%.

In Vitro Results

Among the miH1-miH21 constructs targeting exon 1, miH12 induced thestrongest Luciferase reporter knockdown with a 75-80% reduction (FIG.3A). siRNAs targeting H12, i.e. SEQ ID NO.1, were all shown to havesimilar knockdown efficiency, showing upon an increase in dose astronger knockdown. SiRNAs of 19 and 21 base pairs showed some moreinhibition as compared to the other siRNAs tested. Next generationsequencing (NGS) analysis of the RNA expressed from pVD-CMV-miHTT-155was performed and showed a preference in guide and passenger strands(see e.g. FIG. 2B (7)) corresponding to the first RNA and second RNAsequence as designed to be part of the miRNA scaffold. ThepVDCAG-miH12-451 and

pVD-PGK-miH12-451 were tested for targeting both LucHTT and LucH12_451a.Both the CAG and PGK constructs showed sequence specific inhibition(FIG. 4A), whereas a Luc reporter was comprising a sequence fullycomplementary to the second RNA sequence of the constructs was notreduced.

In Vivo Knockdown Using AAV Vectors in Mice

The expression construct CMV-miH12-155 from pVD-CMV-miHTT-155 was clonedin an AAV5 vector backbone and AAV5 produced using the baculovirusproduction system. As a control CMV-miScr-155 was also incorporated inan AAV5 backbone to serve as a negative control. For monitoring thebrain transduction efficiency, the expression cassettes contained GFP(FIG. 5A). An AAV-5 vector carrying a luciferase reporter construct,Luc73QHTT, comprising the target sequence SEQ ID NO.1 (i.e. the completeHTT exon 1 sequence with 73 CAG repeats) and flanking sequences thereofas present in SEQ ID NO.2 (see FIG. 1B). Balb/6 mice (N5) wereco-injected intrastriatally with 2 μl of AAV5-Luc73QHtt/SNP (3.6×10¹²gc/ml), and AAV5-miScr (1.8×10¹³ gc/ml) or AAV5-miH12 (1.8×10¹³ gc/ml)in a 1:5 ratio. A separate group was injected with AAV5-Luc73QHtt/SNPand PBS. Luciferase expression was monitored at 2, 4, and 6 p.i. byMS.). Already at 1 week p.i., there was a clear knockdown ofLuc19QHtt/wt by miH12 compared to miScr and Luc19QHtt/wt-only animals(FIGS. 7b and c ). A trend was shown indicating a significant decreasein Luciferase reporter expression in time, being almost undetectable(miH12 #1 and #2) in the brain compared with the control groupsindicating a strong knockdown of the HTT target by CMV-miH12-155. At theend of the experiment, the Luc73QHtt/SNP fluorescence in theCMV-miH12-155 group was about 1 log lower compared to miScr.

In Vivo Knockdown Using AAV Vectors in an HD Animal Models

AAV5-CMV-miH12-155 was tested in the LV-171-82Q HD rat model (Drouet etal. 2009, Ann Neurol. 2009 March; 65(3):276-85). The model is based onthe striatal overexpression of the first 171 amino acids of the HTTmutant fragment with 82 CAG repeats linked to a fragment of exon 67containing the SNP C/T. HD rats were injected with AAV5-CMV-miH12-155 ora control AAV5-CMV-miScr-155 (FIG. 6A). Rats were injectedintrastriatally with LV-171-82Q and one week later withAAV5-CMV-miH12-155 or AAV5-CMV-miScr-155. The neuroprotective effect ofAAV5-CMV-miH12-155 was determined based on histological staining(DARP32, EM48, GFP and Iba1) of HD rat brain sections at the early andlate time points (FIGS. 6B, 6C and 6D, 2 weeks p.i.). DARP32 lesions(FIG. 6B, upper panel) indicate neurodegeneration and can be observed aswhite spots on the brain sections. The panel clearly shows less neuronaldeath and hence no white spots in the brain sections ofAAV5CMV-miH12-155 rats. Brain sections were stained for mutant HTTaggregates (EM48, FIG. 6B) seen as small brown dots on the slides. Therewas clearly less neurodegeneration and less mutant HTT aggregates in HDrats injected with AAV5-CMV-miH12-155 as compared to the control group(FIG. 6B, middle and lowest panels). Similar results were obtained atthe late time point of 8 weeks post-injection (data not shown). GFPhistology (brown staining) results indicated efficient striataltransduction with all vectors used in the current study (FIG. 6C).Additionally, almost no immune response was detected at 8 weeks p.i.based on Iba1 staining (FIG. 6D).

AAV5-CMV-miH12-155 was subsequently further tested in the humanizedHu97/18 HD mouse model (Southwell et al. 2013, Hum Mol Genet. 2013 Jan.1; 22(1):18-34). This model has the murine Hdh gene replaced by twocopies of the human HTT, one carrying 97 CAG repeats and the other 18.Detailed characterization of the motor, psychiatric, cognitive,electrophysiological, neuropathological and biochemical changes in theHu97/18 mouse model as a result of disease progression has beenperformed. AAV5-CMV-miH12-155 was injected in 2-months old humanizedHu97/18 HD mouse model by delivery via intracerebral delivery viaconvection-enhanced diffusion (IC-CED) in the striatum or slow (IC-slowdelivery) in the striatum or intracerebroventricular delivery (ICV) inthe ventricles of the brain.

GFP fluorescence indicated complete transduction of the mouse striatumupon slow and CED delivery (FIG. 7A). Western blot analysis of the humanHTT showed knockdown by AAV5-CMV-miH12-155 in the striatum when the CEDdelivery was applied (FIG. 7B).

Comparison H12 with Prior Art Target Sequences

Target sequences from prior art in the proximity of the H12 sequencewere compared with the H12 sequences. siRNAs and miRNA scaffolds wereconstructed and a direct comparison was carried out using the Luciferasereporter system as described above. Low concentrations of siRNAs weretransfected (0.25 nM) in triplicates in order to avoid off-targeteffects skewing the results. The siRNAs were made with fullycomplementary guide and passenger strands (G-C and A-U base pairs) andhaving a UU-3′ overhang in both strands. A scrambled siRNA was used as acontrol and values were measured relative to control. The H12 siRNAshowed strongest inhibition (see FIG. 8A). Likewise, miRNA scaffoldswere made based on miR-155 as described above in accordance with theinstructions of Invitrogen and a direct comparison was made as well. TheR6.1 and R6.2 scaffolds were made by replacing 19 and 18 nucleotidesthat are perfectly complementary to R6.1 and R6.2 target sequences intothe guide sequence of the engineered miR-155 scaffold from Invitrogen.Therefore, depending on the pre-miRNA processing by Dicer, the processedR6 guide strand may contain nucleotides from miR-155 scaffold at theend(s) of the sequence. miRNA scaffolds were transfected as describedabove using different ratios between miRNA construct and reporter (miRNAscaffold construct: Luciferase reporter). A scrambled miRNA constructwas used as a control and values were measured relative to control. FIG.8B shows a 1:1 ratio, whereas FIG. 8C shows a 1:10 ratio. The H12 miRNAscaffold showed strongest inhibition. H12 showed pronounced stronginhibition for both siRNAs and miRNAs, in particular at lowconcentrations which may be considered most relevant for in vivoapplication.

TABLE 4 Targets from prior art compared with H12. Target Target sequenceSI. L. Id. H12 5′-CUUCGAGUCCCUCA 34 21 21 AGUCCUU-3′ H115′-GAAGGCCUUCGAGU 33 21 15 CCCUCAA-3′ R1 5′-GGCCUCGAGUCCCU 46 21 18(siHUNT-2) CAAGUCC-3′ R2 5′-GGCCUUCGAGUCCC 47 21 18 (shD2) UCAAGUC----3′R3 5′-AGGCCUUCGAGUCC 48 21 17 (siRNA- CUCAAGU-3′ DExon1) R45′-AUGAAGGCCUUCGA 49 21 13 (HDAS 07) GUCCCUC-3′ R6.1 5′-GCCUUCGAGUCCCU50 19 17 (54) CAAGU-3′ R6.2 5′-CCUUCGAGUCCCUC 51 18 17 (55) AAGU-3′ Thetarget sequences are shown. The target nucleotides that have identitywith the H12 target sequence are underlined. (SI. indicates SEQ ID NO.;L. indicates the nucleotide length, Id. Indicates the number ofnucleotides that have identity with H12). (R1 is derived from the siRNAsiHUNT-2 from Rodriguez-Lebron et al., 2005, Mol Ther. Vol 12 No.4:618-633, R2 is derived from an expressed shRNA shD2 from Franich et al.,2008, Mol Ther, Vol. 16 No.5; 947-956), R3 is derived from thesiRNA-DExon1 from US20080015158, R4 is derived from HDAS 07WO2008134646, R6.1 and R6.2 are derived from a list of about 1750hypothetical siRNAs designed to target the huntingtin gene(WO2005105995).

The invention claimed is:
 1. A double stranded RNA comprising a firstRNA sequence and a second RNA sequence wherein the first and second RNAsequence are substantially complementary, wherein the first RNA sequencehas a sequence length of at least 19 nucleotides and is fullycomplementarity to SEQ ID NO.
 1. 2. A double stranded RNA according toclaim 1, wherein said double stranded RNA is capable of reducinghuntingtin gene expression.
 3. A double stranded RNA according to claim1, wherein the double stranded RNA is comprised in a pre-miRNA scaffold,a pri-miRNA scaffold, a shRNA, or an siRNA, preferably a pre-miRNAscaffold.
 4. A double stranded RNA according to claim 1, wherein thefirst RNA sequence has a sequence length of at least 20 nucleotides,preferably of at least 21 nucleotides.
 5. A double stranded RNAaccording to claim 1, wherein the first strand is selected from thegroup consisting of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6,and SEQ ID NO.7.
 6. A double stranded RNA according to claim 1, whereinthe first strand and second strand are selected from the groupconsisting of the combinations of SEQ ID NO. 3 and 8; SEQ ID NO. 4 and9; SEQ ID NO. 5 and 10; SEQ ID NO. 5 and 13; SEQ ID NO. 5 and 14; SEQ IDNO. 6 and 11; and SEQ ID NO. and 7 and
 12. 7. A double stranded RNAaccording to claim 1, wherein the double stranded RNA is comprised in apre-miRNA scaffold derived from miR-451a or miR-155.
 8. A DNA sequenceencoding the double stranded RNA according to claim
 1. 9. An expressioncassette encoding the double stranded RNA according to claim
 1. 10. Anexpression cassette according to claim 9, wherein the expressioncassette comprises the PGK promoter, a CMV promoter, a neurospecificpromoter or a CBA promoter.
 11. A gene therapy vector comprising theexpression cassette according to claim
 9. 12. A gene therapy vectoraccording to claim 11, wherein the gene therapy vector is an AAV vector,preferably an AAV vector of serotype
 5. 13. A host cell comprising theDNA sequence according to claim 8 or the expression cassette accordingto claim
 9. 14. A method of treating Huntington's disease comprising,administering to an individual with Huntington's disease the doublestranded RNA according to claim
 1. 15. A method of treating Huntington'sdisease comprising, administering to an individual with Huntington'sdisease the expression cassette according to claim 9.